Project summary Sodium-dependent cation-chloride cotransporters (NaCCCs) have well-described roles in cell volume and intracellular chloride regulation. NaCCCs also contribute to the transport of salt across epithelial cells layers in secretory tissues such as sweat glands, intestine, and lung as well as absorptive tissues such as kidney. Despite their physiological importance and known roles in human diseases including hypertension, salt-wasting diseases, and neurological disorders, many questions remain about their fundamental structure-function properties. For example, we lack sufficient understanding of the structural basis of ion and inhibitor binding by these transporters. Prior studies that compared NaCCCs with different functional properties have been valuable in assessing their structure-function. This project seeks to develop tools to perform comparisons between a group of insect NaCCCs (NaCCC2s) and their mammalian orthologs and paralogs. Such comparisons will offer novel insights into NaCCC structure-function. To accomplish these aims, the NaCCC2s from Drosophila melanogaster and Manduca sexta will be stably expressed in both mammalian (HEK 293) and insect (Sf9) tissue culture cells and their function quantified by flux assays using optical sensors that monitor chloride (ClopHensor derivatives) and pH (pHerry and pHire) and with tracer ion experiments that monitor Li or Rb uptake using cation-chromatography. Expression in oocytes and assessment by ion-selective microprobes will be employed as an alternative and confirmatory approach. Based upon analysis of NaCCC amino acid sequences, we hypothesize that the NaCCC2s have different ion and inhibitor sensitivities and affinities than mammalian transporters and previously characterized insect transporters.